TDP-43 (TAR DNA binding protein of 43 kDa) is a 414 amino acid DNA- and RNA-binding protein that was identified based on its binding to the HIV-1 TAR DNA sequence. While its function is not fully understood, TDP-43 is implicated in transcriptional repression, exon splicing, miRNA generation, cell cycle regulation and apoptosis. The protein contains two RNA recognition motifs (RRM1 and 2) and a C-terminal glycine-rich domain. Ubiquitinated, phosphorylated and insoluble forms of TDP-43 are identified as constituents of inclusions found in TDP-43 proteinopathies that include amyotrophic lateral sclerosis (ALS) and fronto-temporal lobar dementia (FTLD) and Alzheimer's disease (AD) (Arai et al., 2006; Josephs et al., 2014; Neumann et al., 2006).
Wildtype (WT) TDP-43 contains two nuclear localization signals (NLS) in the N-terminal domain of the protein and predominantly localizes to the nucleus; however, truncation mutants lacking the NLS sequences localize to the cytoplasm (Ayala et al., 2008; Winton et al., 2008). Proteolytic fragments (e.g. C25) and higher molecular weight species of TDP-43, including soluble oligomers and insoluble aggregates, localize to the cytoplasm and are associated with toxicity. The accumulation of TDP-43 leads to a loss of cellular function, thereby impairing the viability of the affected nerve cells. Disease associated point mutations in TDP-43, of which 32 have been identified, are primarily located in the C-terminal domain (Pesiridis et al., 2009). While the role of these mutations in causing disease is not fully understood, pathological forms of TDP-43 identified in AD, ALS and FTLD include higher molecular weight, aggregated TDP-43, which is often phosphorylated and ubiquitinated.
In the AD brain TDP-43 pathology can present as cytoplasmic and intranuclear inclusions in neurons. TDP-43 pathology in AD is associated with an increased risk for hippocampal atrophy, memory loss and cognitive impairment. Recent data from another group suggests that TDP-43 plays a key role in AD pathogenesis and that people with TDP-43 pathology were at a greater risk for hippocampal atrophy, memory loss and cognitive impairment.
It has recently been suggested that TDP-43 oligomers, aside from filamentous aggregates, may play a role in TDP-43 pathogenesis. It was found that recombinant full-length human TDP-43 forms structurally stable, spherical oligomers that share common epitopes with an anti-amyloid oligomer-specific antibody. The TDP-43 oligomers are stable, have exposed hydrophobic surfaces, exhibit reduced DNA binding capability and are neurotoxic in vitro and in vivo. Moreover, TDP-43 oligomers are capable of cross-seeding Alzheimer's amyloid-beta to form amyloid oligomers, demonstrating interconvertibility between the amyloid species. Such oligomers are present in the forebrain of transgenic TDP-43 mice and FTLD-TDP patients. (Fang, Y S et al., Full length TDP-43 forms toxic amyloid oligomers that are present in the frontotemporal lobar dementia-TDP patients, Nature Communications, (2014), 5:4824)
There is some evidence that the C-terminus of TDP-43 shows sequence similarity to prion proteins which raises the possibility that TDP-43 derivatives may cause spreading of the disease phenotype among neighboring neurons. Synthetic peptides flanking residue 315 form amyloid fibrils in vitro and cause neuronal death in primary cultures. Given the biochemical similarities between TDP-43 and prion proteins, there may be therapeutic potential for decreasing the abundance of neurotoxic TDP-43 species, enhancing degradation or clearance of such TDP-43 derivatives and blocking the spread of the disease phenotype. (Guo, W. et al., An ALS-associated mutation affecting TDP-43 enhances protein aggregation, fibril formation and neurotoxicity, Nature Structural & Molecular Biology, 2011, 18:822-830)
ALS is a fatal progressive degeneration of motor neurons in the brain and spinal cord. Whereas 90% to 95% of ALS cases are sporadic, gene mutations in copper/zinc superoxide dismutase 1, senataxin, dynactin, angiogenic, and TAR-DBP (the gene for transactive response DNA-binding protein of 43 kd [TDP-43] on chromosome 1) account for some familial forms of the disease. Although TDP-43 was originally thought to be a specific marker for ALS and frontotemporal lobar degeneration (FTLD) with tau-negative ubiquitin-positive TDP-43-positive inclusions (FTLD-U, recently renamed FTLD-TDP), TDP-43-positive inclusions have now been found in a variety of other neurodegenerative disorders. Diseases with secondary TDP-43 pathology include AD and hippocampal sclerosis, Guam parkinsonism-dementia complex (PDC), Pick disease, corticobasal degeneration, argyrophilic grain disease, and Lewy body disease. (McKee, A. et al., TDP-43 proteinopathy and motor neuron disease in chronic traumatic encephalopathy, J Neuropathol Exp Neurol., 2010, 69(9):918-929)
It has been suggested that repetitive head injury is associated with the development of chronic traumatic encephalopathy (CTE). Patients suffering from CTE have recently been shown to have TDP-43 proteinopathy affecting the frontal and temporal cortices, medial temporal lobe, basal ganglia, diencephalon and brainstem. The TDP-43 proteinopathy associated with CTE is similar to that found in frontotemporal lobar degeneration with TDP-43 inclusions, in that widespread regions of the brain are affected. Akin to frontotemporal lobar degeneration with TDP-43 inclusions, in some individuals with CTE, the TDP-43 proteinopathy extends to involve the spinal cord and is associated with motor neuron disease. It is thought that traumatic axonal injury may also accelerate TDP-43 accumulation, aggregation, and dislocation to the cytoplasm and thereby enhance its neurotoxicity. (McKee 2010)
Hexachlorophene (also referred to herein as B10), an organochlorine compound, was widely used as an effective antiseptic for topical applications until the 1970s (Kimbrough, 1971; Pilapil, 1966). It has been found to cause toxicity in animal models (Kimbrough and Gaines, 1971; Thorpe, 1967), and its use was discontinued due to the severity of side effects in human beings. It is generally prepared by the condensation of 2 moles of 2,4,5-trichlorophenol with 1 mole formaldehyde in the presence of concentrated sulfuric acid. B10 can be absorbed into the body through the skin or by ingestion. Oral toxicity studies in rats show an LD50=66 mg/kg.
It has been shown to inhibit the Wnt-β-catenin signaling pathway in B lymphoma cells (Min et al., 2009), and recently to inhibit amyloid beta (Aβ) fibril formation and protect primary neuronal cultures from Aβ-induced toxicity (Eleuteri et al., 2014).
In overcoming the toxicity of hexachlorophene (B10) administration alone, another group found that pre-treatment with antioxidants, such as butylated hydroxytoluene (BHT) and ethoxyquin, prior to administration of B10 protects rats from toxicity induced by B10. BHT and ethoxyquin are both free-radical scavengers that prevent lipid peroxidation. In addition, this group also found that agents, such as phenobarbital and SKF-525A (2-diethylaminoethyl-2,2-diphenylvalerate-Hcl), also lessened toxicity when administered prior to B10. The pre-treatment agents were administered daily for at least 3 days prior to administration of B10. It was noteworthy that the protective effect of SKF-525A only occurred upon oral administration of the drug, not ip administration. It was suggested that this effect may occur because a higher SKF-525A is a cytochrome P450 inhibitor which is known to potentiate barbiturate effects and phenobarbital is a barbiturate itself (Hanig, J. P. et al., Protection with butylated hydroxytoluene and other compounds against intoxication and mortality caused by hexachlorophene, Fd Chem. Toxic., 22(3):185-189)
A different group also noted that pre-treatment with the antioxidant L-carnitine also protected rats from the toxicity of B10. (Yapar, K. et al., Protective effects of L-carnitine on the hexachlorophene-induced neurotoxicity and oxidative stress in mice, Revuede Medecine Veterinaire, 2007, 158(12):607-612)
Given the limited options that exist for treatment of TDP-43 proteinopathies, what is needed is a drug that is capable of reducing TDP-43 expression in patients suffering from TDP-43 proteinopathies. It would be advantageous to identify pharmacological agents that may inhibit the formation of TDP-43 aggregates or aid in the clearance of such aggregates from cells.